1. Field of the Invention
The present invention relates to the field of Immunology, specifically with the branch of Immuno-Allergy and in particular with the use of adjuvant or carrier compounds, capable of modulating the immune response to allergens. The aim is to induce changes in this specific response that can reduce or prevent its pathogenic effect.
Allergy is a very frequent pathology, mainly in the industrialized countries. In allergy intervenes several immunopathological mechanisms and particularly the so-called type I anaphylactic hypersensitivity. Physiopathogeny of type I hypersensitivity is based on the increased production of IgE, a cytotropic antibody. The IgE antibodies are raised against foreign substances, denominated allergens, after a first exposure, denominated sensitization.
IgE antibodies can bind to the IgE receptors on the surface of blood basophiles and tissue mast cells, prolonging their mean life from a few hours in blood to several months in tissues. Later contacts of the allergens with the cell-bound IgE antibodies can provoke the cross-linking of neighbor antibodies, which trigger a signal cascade to the cell cytoplasm, leading to cell degranulation and release of inflammatory mediators such as histamine, serotonine and kinines
The most common allergic diseases are rhinitis, asthma, and atopic dermatitis. Allergic asthma is a chronic inflammatory disorder. In the immunopathogenesis of asthma are involved not only IgE dependent mechanisms (responsible for the Type 1 anaphylactic response), but also T-cells, and particularly the so-called Th2 cell subset. Th2 lymphocytes induce and maintain the inflammatory response by means of the recruitment and activation of other cells and particularly through IL-5 production, which is responsible for the recruitment and activation of Eosinophiles. Eosinophilia plays an important role in the development of bronchial hyperreactivity, which is an essential feature of asthma.
Common allergens are pollen, house dust mites, moulds, drugs, foods and animal hair and dander. Indoor allergens and specially, house dust mites, are a very relevant cause of respiratory allergy and asthma.
Clinical manifestations of allergic disorders are usually treated with anti-histaminics, β-agonists, sodium chromoglicate and corticosteroids. These treatments are generally inadequate, because they are purely symptomatic and do not influence the ethiopathogeny of the disease. Nowadays, it is widely recognized the need of more effective treatment approaches, among them, specific immunotherapy or therapeutic vaccines.
2. Background of the Prior Art
Traditionally, the hyposensitization therapy with allergen extracts (also called specific immunotherapy) has been widely used in the treatment of allergic diseases. In this type of treatment, the patients are administered with periodical subcutaneous injections of the specific offending allergens. It is started injecting small allergen doses and if allergic reactions do not appear, the dose is increased. The injections are administered with a weekly frequency at the beginning; then, doses are gradually increased until reaching a maintenance dose at monthly or semimonthly frequency. This treatment should be maintained for several years (WHO Position Paper. Allergen Immunotherapy: Therapeutic Vaccines against Allergic Diseases. Geneva, January 1998).
In spite of the method is regarded as relatively effective for some allergic diseases, the immunotherapy has been questioned due to safety reasons. During this treatment, the patient can suffer from severe anaphylactic reactions, which eventually can be even fatal. In addition, the high number of injections to administer, that can be about 100-200 in 3 years, constitutes a serious drawback for its practical application. Poor patient compliance and premature abandonment of the treatment are frequent causes of lack of expected effectiveness.
In recent years, it has been a great progress in the understanding of the mechanisms of the induction of IgE immune response in allergic subjects. IgE production by B-cells is driven by specific mechanisms of the Th2 immune cell response, and particularly, it is induced by an overproduction of IL-4 by Th2 cells. The Th2 cells are involved pathologically not only through their regulatory role (IL-4 induces the class switch to IgE in B-lymphocytes, mediated by a contact signal provided by T-cells), but also participate directly in the effector phase of the delayed type allergic inflammatory response and in the asthmatic chronic inflammation.
The exposure to environmental allergens, in addition to the own genetic predisposition, constitutes the leading phenomenon of the allergic sensitization process and causes the triggering of allergic reactions.
The immunological mechanism responsible for clinical improvement of the patients during allergen immunotherapy is not totally explained yet. Nevertheless, it is known that several immunological changes take place, linked to a long-term decrease of allergen specific IgE: there is a decrease of IL-4 and IL-5 secretion and increase of IFN-γ and IgG levels. These changes indicate a decrease of the Th2 response pattern, like a possible consequence of the induction of a non-pathogenic concurrent Th1 pattern (WHO Position Paper. Allergen Immunotherapy: Therapeutic Vaccines against Allergic Diseases. Geneva, January 1998).
The Th1/Th2 response patterns distinguish themselves firstly by the typical pattern of cytokines, secreted by Th-cells: namely IFN-γ and IL-2 by Th1 and IL-4 and IL-5 by Th2 (Mossman, T. R., Cherwinski, H., Bond, M. W., Giedlin, M. To, and R. L. Coffman. 1986. Two types of murine T helper cell clones. I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 136:2348-2357). On the other hand, it is also possible to determine the type of induced response determining serum Ig class/subclass profiles. Thus, murine Th1 pattern induces antibodies preferably of the IgG2 subclass (IFN-γ dependent), whereas Th2 induces IgE and IgG1 subclass (IL-4 dependent). In humans, Th1 is linked to IgG1/IgG3 antibodies and Th2 to IgE.
Although the onset and development of Allergy are caused by environmental factors, the fundamental role in the propensity of an individual to develop a Th2/IgE response is of genetic order (Holgate ST. Environmental Genetic and interaction in allergy and Asthma. J Allergy Clin Immunol 1999; 104:1139-1146). Therefore, the descendants of allergic parents will develop an allergic disease with a greater frequency than the children of non-allergic parents. This frequency has been estimated as 75, 50 and 25% if both parents, the mother or the father are allergic, respectively). That means that applying a diagnostic system to the parents, the behavior of an individual within the population, can be predicted with a high likelihood. Additionally, it is well known that the establishment of a pathological Th2 pattern occurs in the first 6-24 months of life. Environmental factors like exposition to bacterial infections can influence positively on the disease onset, inducing a Th1 pattern, which down-regulates the allergic Th2 pattern (Holt PG, Programming of Allergen Specific Th-memory during Childhood. Proceedings XVII International Congress of Allergology and Clinical Immunology. Sydney 2000. Allergy Clin Immunol International Suppl. 1, 2000 pp 83-85). These findings suggest the possibility of designing prophylactic anti-allergic vaccines, which could prevent the establishment of a Th2 response to allergens, in the early childhood. These vaccines could be based on Th1 adjuvants, which ideally should be effective during the breast-feeding period.
Several approaches have been attempted to improve allergen immunotherapy, aiming to reduce their negative aspects, preserving or extending their benefits. Particularly, it is known that several methods have been used in order to modify the allergens so that its allergenicity (i.e., its ability to be recognized by IgE antibodies) and ability to induce IgE antibodies following immunization could be reduced, as well as, to increase their immunogenicity (i.e. its capacity to induce a therapeutic or possibly protective response). Among them, there are physical methods by adsorption of allergens, creating a depot effect (adjuvants), or chemical methods aiming at modifying allergen molecules by means of covalent links with other compounds or between themselves.
Several physical agents have been used, among them: Tyrosine (Patent No. GB 1,377,074), Tyrosine Ester (U.S. Pat. No. 4,428,932); Liposomes (Patent Application WO 89/10753); Monophosforil Lipid A (MLA) (U.S. Pat. No. 5,762,943); Saponine (U.S. Pat. No. 4,432,969) and the traditional ones: Aluminum Hydroxide and Calcium Phosphate.
Among the chemical agents that have been used for allergen polymerization, are Formaldehyde (Marsch DG et al. Studies on allergoids from naturally occurring allergens. III Preparation of Ragweed pollen allergoids by aldehyde modification. J. Allergy Clin. Immunol. 1981; 68:449-59); Alginate (Corrado O J et al. Allergy 1989; 44:108-5) and Glutaraldehyde (Patent No. GB 1,282,163). Also, there have been used: MPEG (Dreborg et al. Crit Rev Ther Drug Carrier Syst. 1990; 6:315-65) and polysaccharide conjugates (European Patent Application No. EP 3497 008 A2).
These solutions could, in some cases (Eg. Glutaraldehyde polymerized allergens, Alum adjuvanted, Grammer et al., Modified forms of allergen immunotherapy, J Allergy Clin Immunol 1985; 44:108-15) increase the allergen-specific IgG titters in animal models. Nevertheless, the increase of this antibody class does not necessarily imply a clinical improvement of the patient, mainly if a parallel increase of IgE antibodies occurs. In fact, some IgG subclasses (IgG1 in mice and IgG4 in humans) are involved, also, in different allergic reactions, due to its ability to bind themselves, transitorily, to mast cell surface. On the other hand, clinical evidences in humans indicate that the increase of IgG antibodies is not correlated directly with clinical improvement, suggesting that these antibodies play only a secondary role in the regulatory mechanism, induced by the treatment (Rak S. Lowhagen O., Venge P. Bronchial The effect of immunotherapy on hyperresponsiveness and eosinophil cationic protein in pollen-allergic patients. J Allergy Clin Immunol 1988; 82:470-80 and Jutel M, Müller Or, Fricker M, Rihs S, Pichler W, Dahinden C. Influence of bee venom immunotherapy on degranulation and leukotriene generation in human blood basophiles. Clin Exp Immunol 1996; 12:1112-18)
Although, chemically modified allergens (so-called allergoids), has some advantages concerning the decrease of secondary adverse reactions during the treatment as compared to traditional allergen extracts, they do not produce a significant clinical improvement (Bousquet et al. J. Allergy Clin. Immunol. 1989; 84:546-56 and Grammer et al. J Allergy Clin Immunology 1985; 76:397-401). Another drawback of allergoids, used so far in clinical trials (modified by Glutaraldehyde or Formaldehyde), is the practical impossibility to obtain a standardized composition, since the final products are obtained by means of chemical reactions starting from allergen extracts, which are heterogeneous mixtures of polypeptides and other biomolecules. In these reactions, it cannot be avoided that molecular species with different degrees of polymerization or chemical modifications are formed, which can constitute unwanted, difficult to eliminate by-products.
The use of Aluminum Hydroxide has only managed to reduce the number of injections necessary to obtain the same effect that with the non-adjuvanted allergens, which is attributed to its depot and slow release effects. Nevertheless, it is known that this adjuvant stimulates IgE production, as much in animals as in humans, inducing a typical Th2 pattern, which could possibly reinforce the pathogenic allergic response, during its administration.
Calcium Phosphate and Tyrosine have also been used as adsorbents for creating a depot effect and slow release of the allergens. Nevertheless, these formulations have shown no significant advantages as compared to aqueous or Alum-adjuvanted extracts, regarding the effectiveness or safety of the treatment (Altintas D U et al. Comparison between the use of adsorbed and aqueous immunotherapy material in Dermatophagoides pteronyssinus sensitive asthmatic patients. Allergologie et Immunopathologie 1999; 27(6):309-317).
None of the existing at the present time, previously mentioned solutions could achieve completely effective immune modulation (expressed in inhibition or reduction of Th2 pattern or induction of Th1) neither in animals nor in humans, and no clinical effectiveness has been shown in patients, using a reduced number of injections.
The use of liposomes containing allergens has been described in the Patent Application WO 89/10753. It is regarded that liposomes, besides creating a depot and reducing allergenicity of the product, can influence the mechanisms of antigen presentation to the immune system, due to their lipidic and particulate nature (WHO Position Paper. Allergen Immunotherapy: Therapeutic Vaccines against Allergic Diseases. Geneva, January 1998). Nevertheless, this approach has several drawbacks that have hampered a wide introduction into the clinical practice. Among them: the lack of stability of these formulations and availability of a technology for inclusion of allergens into liposomes in a consistent way. The lack of stability of the liposome formulation during storage or administration to the patient has serious implications in the safety of the treatment, since the possible uncontrolled release of allergens from liposomes can cause severe anaphylactic reactions, similarly to allergens in aqueous form. Moreover, definitive evidences of the induction of an effective immune response, either that affects Th1/Th2 balance or that induces a non-pathological or protectogenic pattern, have not been obtained.
More recent approaches have tried to use allergen formulations with adjuvant inducers of Th1 response, such as Monophosforil Lipid A (MLA) (U.S. Pat. No. 5,762,943) and Heat Shock Proteins (HSP) also known as Mycobacterium Stress Proteins, which are described in the patent WO9823735. MLA, a detoxified variant of LPS, has been shown that reduces specific IgE and increases IgG levels, in experimental mice models. Nevertheless, it has not been demonstrated that it can effectively reduce the Th2 cellular component of the allergic response. Therefore, its use would be limited strictly to the allergic diseases for which the type 1 hypersensitivity mechanism is the main one, which would exclude asthma. Another major drawback is that, in spite of the fact that the toxicity of LPS has been attenuated, it has not been completely removed (Baldrick P et al. Vaccine 20, 2002 737-743), which could limit its use in small children. In the case of HSP, experimental evidences have not been provided, supporting that clinically relevant allergens mixed or conjugated to these proteins could produce a deviation of allergen-specific Th2 response, neither in animal models nor in humans.
The use of proteoliposomes derived from the outer membrane of Gram-negative bacteria has described for prophylactic vaccine formulations against infectious diseases, by Ruegg C L and cols. (Preparation of proteosome-based vaccines. J Immunological Methods 1990; 135:101-9); Lowell and cols. (Proteosome-Lipopeptide Vaccines Enhancement of Immunity for Malaria CS Peptides. Science 1988; 240:800-2); also in the U.S. Pat. No. 5,597,572. In this last case, the main core is an outer membrane proteoliposome or vesicle (OMV) derived from Neisseria meningitidis serogroup B. It is regarded that its particulate structure, lipo-oligosaccharide traces (LPS) incorporated into the OMV; polysaccharide C; lipid composition and adsorption into Alum, are relevant for its proven immunogenicity and protectogenicity in humans.
In spite of containing LPS, this vaccine formulation does not cause toxic effects neither in humans nor in animals, which is attributed to the OMV peculiar structure and composition. On the other hand, the immunomodulator and adjuvant effect of LPS could be retained.
The anti-meningococcal vaccine, based on this proteoliposome, has been successfully applied in more than 50 million doses, demonstrating to be safe, non-reactogenic and effective to protect against N. meningitidis serogroups B and C. Moreover, it can be applied safely during the breast-feeding period. It has been shown that it is able to turn the T-independent Polysaccharide C antigen into a T-dependent one. This vaccine induces a preferential Th1 pattern in humans and animals, characterized by the induction of lymphoproliferation; anti-OMV IgG antibodies (subclass IgG1 in humans and IgG2a in mice); IFN-γ, IL-2 and IL-12, both at protein and mRNA level. It does not induce anti-OMV IgE, neither increases total IgE levels, nor IL-4, IL-5; both, at protein or mRNA level (Infect Immun. 2001, 69(72001):4502-4508). The response induced by the vaccine, as it is usual for the adaptive immunity, is specific to the bacterial antigens contained in the product, and therefore, there are no evidences of induction of an immune response towards well-known common allergens.